Project Summary The hepatitis C virus chronically infects ~3% of the worldwide population, and is responsible for the majority of liver cancers and transplantations in the Western Hemisphere. Chronic HCV infections persist for decades, likely by continuously spreading to new hepatocytes, despite an active immune response by the host. During infection, HCV enters hepatocytes through a complicated multi-step entry process. Indeed, our long-term goal is to use HCV as a model to better understand multistep mechanisms of viral entry into host cells. The proposed experiments are based on the hypothesis that the HCV cell entry process represents a pathway, where virions directly or indirectly utilize host factors in a sequential manner to be trafficked to an endosome in a fusion competent form. This hypothesis is based upon the observations that: i) targeting HCV entry factors by blocking antibodies or RNAi impairs distinct stages of HCV cell entry; ii) HCV virions require incubation with host cells to be primed for low pH-dependent fusion; and iii) HCV glycoprotein mutations can alter host entry factor dependency. Although numerous host factors are implicated in mediating HCV host cell entry, how these factors actually mediate HCV cell entry has not been determined. In this application, I propose to use CRISPR to completely knock out the genes for host factors that have been previously implicated in playing a role in HCV cell entry. I will first characterize the capacity for HCV to enter these knockout cells (Aim 1). Preliminary results show that this approach permits the accurate definition of host factor requirements, ultimately classifying these factors as either essential or non-essential HCV host cell entry factors. I will next investigate the mechanisms of action of host entry factors through biochemical, imaging, and molecular tools (Aim 2). For instance, using an assay testing the capacity to induce HCV viral fusion at the cell surface revealed that numerous host factors are essential for priming the HCV fusion protein, which suggests that this priming event occurs late in the entry process. Finally, I will use genetic approaches to understand how viral and host entry factors interact (Aim 3). The information from these experiments is expected to more clearly define the mechanisms of viral entry during initial and chronic HCV infections. HCV is currently a model of multi-step host cell entry, and a deeper understanding of its entry mechanisms will benefit studies of other viruses with multiple receptors or entry steps. Furthermore, understanding the mechanisms of viral entry will inform ongoing efforts to develop an effective HCV vaccine as eliciting neutralizing antibodies that block this stage of the viral life cycle are likely to be an important component of such a vaccine.